Threshold outputs of gene expression are important for patterning all metazoa. One of the central challenges in the field of developmental biology in this post-genomics era is to determine how large numbers of genes are differentially expressed to pattern the embryo and how these genes subsequently control differentiation of specialized tissues. Whole-genome approaches to studying development typically generate many lists of genes that are predicted to function in regulating biology. We propose to utilize genomic data to study transcriptional regulation of development in Drosophila and relate the mechanisms uncovered in this system to humans. Specifically, we are interested in how dorsoventral patterning of the Drosophila embryo is controlled by the Dorsal-dependent morphogen gradient. By microarray analysis, we have found that ~50 genes are transcriptionally regulated by Dorsal and are differentially expressed along the dorsoventral axis in distinct domains. Several of the identified genes encode transcription factors that may function together with Dorsal to control patterning of gene expression along the dorsoventral axis. Our initial results on Dorsal- dependent target gene expression also raise a number of questions regarding threshold outputs of gene expression. In particular, how is the expression of Dorsal target genes spatially restricted to distinct domains along the dorsoventral axis and do transcription factors that were identified in the microarray screen function together with Dorsal to control patterning? We have proposed two specific aims to address these questions: (1) to identify transcription factors that function together with Dorsal through enhancer analyses (using cis- regulatory motifs we have identified are important for expression), and (2) to characterize the function of two putative transcription factors which we propose contribute to patterning of the embryo (Neu2, identified by our microarray studies, and Wek, a maternally expressed gene identified in a genetic screen). Characterization of the mechanisms used by these transcription factors to affect differential gene expression n distinct domains will provide generally applicable insights into the regulation of gene expression controlled Dy morphogen gradients. These studies may also provide insights into transcriptional regulation by Rel transcription factors in general, and therefore have the potential to provide far-reaching implications into processes regulated by NFkappaB including the immune response, cancer, and allergic inflammation .